The present invention relates to novel derivatives of procainamide and N-acetylprocainamide (NAPA). The derivatives include immunogens used to stimulate antibody production and polypeptide conjugates useful in immunoassays for detecting procainamide and NAPA. Also provided are hapten intermediates in the synthesis of the immunogens and polypeptide conjugates.
The cardiac depressant drugs procainamide and N-acetylprocainamide are used clinically to treat or prevent cardiac arrhythmia. N-acetylprocainamide (abbreviated as NAPA and also known as acecainide) is the major metabolite of procainamide in man. The concentration of this metabolite in the plasma of patients receiving procainamide often exceeds the concentration of the parent drug itself. Metabolism is by in vivo acetylation, and much genetic-based variation has been observed in the rate at which individual patients transform the drug to its metabolite. This phenomenon is of importance in the clinical use of the drugs because of the lower incidence of side effects associated with NAPA. Both the therapeutic usefulness and the toxicity of the drugs are better correlated with their blood levels than with their dosages. The relationship between the amount of drug administered and the blood levels is quite variable. It is influenced by completeness of absorption, distribution characteristics and rates of metabolism and excretion.
Because of these considerations, numerous analytical methods have been developed to determine blood levels of these drugs, including high pressure liquid chromatography (HPLC), quantitative thin layer chromatography (TLC), and immunoassay, including enzyme immunoassay and immunoassay using fluorescence techniques. Competitive binding immunoassays have proved to be particularly advantageous. In such assays, an analyte in a biological sample competes with a labeled reagent, or analyte analog, or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes, fluorescent molecules, and radioactive compounds are common labeling substances used as tracers. The concentration of analyte in the sample determines the amount of analyte analog which binds to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.
One type of competitive binding immunoassay is based upon the reassociation of polypeptide fragments to form active enzymes as a step of generating a detectable signal utilized to determine the amount of analyte present in a sample. This type of assay, known as cloned enzyme donor immunoassay (CEDIA), is described in U.S. Pat. No. 4,708,929, the content of which is herein incorporated by reference. In particular, a .beta.-galactosidase enzyme donor polypeptide combines with a .beta.-galactosidase enzyme acceptor polypeptide to form active .beta.-galactosidase enzyme. Conjugating a hapten, or a small analyte or an analyte analogue, to the enzyme donor polypeptide at certain sites does not affect the ability to form .beta.-galactosidase by a complementation reaction and hence does not affect the rate of .beta.-galactosidase activity when in the presence of a substrate for .beta.-galactosidase. However, when the enzyme donor-hapten conjugate is bound by anti-analyte antibody, the complementation rate is impeded, and thereby the enzyme-catalyzed reaction rate during the initial phase of the reaction is reduced. This reduction in enzyme-catalyzed reaction rate can be monitored and has been used to quantitate the determination of a plurality of analytes on the principle of competitive inhibition where enzyme donor-analyte conjugate present in a reaction mixture and analyte present in the sample compete for anti-analyte antibody prior to the addition of enzyme acceptor. The complementation rate of .beta.-galactosidase formation, and hence enzyme catalyzed reaction rate, is increased as the amount of analyte present in the sample is increased.
In accepted clinical practice, procainamide and NAPA are analyzed separately. Therefore immunoassays for NAPA and procainamide require antibodies with a high degree of specificity for either the metabolite or the drug. Since the metabolite and drug differ only by the presence or absence of an acetyl function, the generation of specific antibodies is a particularly challenging problem. Surprisingly, however, the immunogens of the present invention have been found to be especially useful for this purpose.
The preparation of antibodies to procainamide and NAPA for use in immunoassays to determine the drugs has been accomplished in the prior art by essentially three different approaches. One approach has been to couple procainamide through the benzene ring amino group by diazotization and subsequent condensation to an albumin carrier [A. S. Russel et al., Clin. Exp. Immunol. 3:901 (1968) and Mojaverian et al., J. Pharm. Sci. 69:721 (1980)]. The resulting antibodies show a high degree of cross-reactivity with NAPA, however, and are therefore unsuitable for use in immunoassays specific for one or the other drug.
The second approach involves coupling of the drugs at the opposite end of their structures, at the N-diethylamino group, by modification of one of the ethyl substituents for subsequent coupling to an antigenic carrier. As a result, antibodies are raised against an immunogen in which a major functional group of the drugs has been modified in order to couple them to the carrier. An example of this approach is described in U.S. Pat. No. 4,235,969 issued to Singh et al., in which one of the N-alkyl groups is replaced with a nonoxocarbonyl-alkyl substituent. The nonoxocarbonyl functionality, a linking group containing a carbonyl or imino function, is employed for conjugation to antigens and enzymes. Similarly, European Appl. No. 86103161.5 (Heiman et al.) discloses antigenic conjugates and enzyme conjugates of procainamide analogs modified at the terminal diethylamino group. A specific linking group is attached to a poly(amino acid) or a fluorescein tracer.
In a third approach, Buckler et al., U.S. Pat. No. 4,673,763, describe derivatives of procainamide or NAPA coupled at the .alpha.-position of the amide side chain to an immunogenic carrier material or to a label.
The present invention differs from the prior art in the substitution of a non-cyclic diethylamino group with a cyclic piperidine carboxylic acid group. Conjugation to antigens and enzymes is then accomplished through the carboxyl group, either directly or with crosslinkers. In the conjugates of the present invention, piperidine carboxylic acid modified haptens are preferably reacted with aminoalkyl-maleimide crosslinkers to give maleimide adducts of piperidine carboxylic acid haptens. The latter derivatives are reacted with sulfhydryl groups on carrier proteins, enzymes or enzyme donor polypeptides to give thioether linked hapten conjugates. Maleimide/sulfhydryl chemistry [M. Brinkley, Bioconjugate Chem. 3:5 (1992)] is more easily controlled than amide bond condensation, thus allowing the preparation of immunogens and enzyme or enzyme donor conjugates with defined, targeted degrees of substitution, a feature which is very important to the functional efficacy of the conjugates. Another example of maleimide derivatives and conjugates of procainamide and NAPA are those described in copending U.S. Ser. No. 08/169,851, filed Dec. 17, 1993, the content of which is incorporated herein by reference.